The pyrogenic exotoxins of Group A streptococci and the enterotoxins of Staphylococcus aureus, which are also pyrogenic exotoxins, constitute a family of structurally related toxins which share similar biological activities (11, 13). The staphylococcal and streptococcal pyrogenic exotoxins also share significant amino acid homology throughout their sequences (11, 19, 40). This pyrogenic exotoxin family contains nine main toxin types, and several allelic variants (subtypes) have been described. Several studies have shown that the toxins share common motifs based on immunologic cross reactivity between the toxins (26, 27). They stimulate CD4+, CD8+ and γδ+ T cells by a unique mechanism. These toxins share the ability to bind the β chain variable region (Vβ) elements on the lateral face of the T cell receptor (TCR) and simultaneously bind to the lateral face of the class II major histocompatibility complex (MHC) of antigen presenting cells (FIG. 1), causing an aberrant proliferation of specific T-cell subsets (3, 4, 12). This property of the toxins has labeled them as “superantigens” (36) since they do not interact with the MHC and TCR molecules in the manner of conventional antigens (14, 18) and produce a massive proliferation of T cells.
The variability of the sequences in the TCR-binding region and within the MHC-II-binding regions most likely provides the different suerantigen toxins their specificities for different Vβ molecules and variable affinities for MHC-II types. (69–70)
The cross-linking of TCR with MHC-II molecules by superantigens causes a profound blastogenesis of lymphocytes and antigen-presenting cells. The resulting stimulation of leukocytes leads to a significant increase in cytokine production.
Monocytes stimulated with bacterial superantigens produce the Th1 cytokines IL-2 and IFN-γ and the anti-inflammatory cytokines IL-10 and IL-1 receptor antagonist. (71). T cells activated by superantigen stimulation produce IL-12. (72). Whole preparations of peripheral blood mononuclear cells containing lymphocytes and antigen-presenting cells elicited a wide range of inflammatory cytokines in significant amounts. The generation of monocyte cytokines such as IL-1, IL-6, TNF-α, and TNF-β was dependent on the presence of T cells. (73).
Costimulatory molecules important in conventional immune responses also play a significant role in the response of immune cells to superantigens. The costimulatory T cell antigen, CD28, and its corresponding ligand on MHC-II-bearing cells, B7, contribute to superantigen mitogenicity. (74, 75). Other costimulatory molecules, such as LFA-1/ICAM-1 and VLA-4/VCAM-1, also contribute to the activation of immune cells by superantigens. (76, 77). These immunostimulatory activities of superantigens are crucial to their ability to cause injury to the host.
The bacterial toxins cause a variety of syndromes in humans. Staphylococcal enterotoxins have been implicated in staphylococcal food poisoning (26), as well as toxic shock like syndromes (1). The gene sequences and deduced amino acid sequences of at least six staphylococcal enterotoxins (“SE”): A, B, C, D, E and H, are known, i.e., SEA, SEB, SEC, SED, SEE, and SEH (19, 23). The streptococcal pyrogenic exotoxins (“SPE”) have been implicated in causing the symptoms of scarlet fever and toxic shock like syndrome (8, 20, 30). The sequences of three members of this family are known: SPEA, SPEC, and SSA (5, 23, 35).
Toxic shock syndrome toxin (TSST-1) from S. aureus shares similar biological activity with the SE's and SPE's, however amino acid sequences of this toxin are significantly different from these two classes of toxins (2). Structural analysis suggests that, despite the differences in amino acid composition, the overall topology of TSST-1 and the SE/SPE family of toxins is similar (41). The molecular structure of SE's and SPE's has been determined by various methods. Reviews concerning the molecular structures are available (19, 62). Molecular evolution studies of the SE/SPE family of toxins suggests that the toxins can be grouped into two main clades(34). All these toxins are highly resistant to denaturation by heat and to proteases. With the exception of TSST-1, they are soluble proteins of approximately 230 amino acids and have a central disulphide loop. In contrast TSST-1 has only 194 amino acids and does not possess any cysteines.
It has been suggested that the conservation of amino acids is important to maintaining the structure necessary for the biological activity of the toxins (32). Mutations constructed in various positions throughout the SPEA and SEB molecules were sufficient to inactivate biological activity (6, 15). Mutations at various points throughout the molecules often had different effects, suggesting that functional activities could not be attributed to any one region of the toxins (7). These results suggested that a functional tertiary structure must be maintained. Chemical modifications of highly conserved histidine residues inactivated biologic activity (29). The high conservation of the disulfide loop in the SE's and SPE's suggests an important role in the structure of the SE/SPE family of toxins. Studies show the disulfide loop is required for mitogenic activity of SEA and SEB. Reduction of the disulfide loop inactivated T cell stimulatory activity, but did not affect MHC-II binding and stimulation of monocytes (54). Peptide cleavages within the loop had no effect on T-cell mitogenicity, however cleavage of conserved sequences outside the loop of SEA resulted in loss of mitogenic activity. The loop and conserved adjacent sequences appear to be associated with avidity of the toxins to the TCR, and do not contribute to the specificity of toxins for a particular Vβ type (6). Residues determining TCR Vβ specificity appear to be located within the carboxy-terminus of the SE/SPE toxins (59), while residues critical for MHC-II binding appear to be located in the amino-terminal region, and the central portion of the molecule near the disulfide loop (53). The disulfide loop and adjacent highly conserved sequences contribute to the structural integrity of the toxins, and serve to bring the TCR and MHC binding regions in functional proximity to each other (65).
The SEs are named for their ability to induce gastrointestinal illnesses upon oral intake of a few micrograms of the toxin. The clinical effect appears in 2 to 4 hours and is manifested by nausea and diarrhea. These symptoms appear to be caused by leukotrienes and histamine released from mast cells. Additionally, both the staphylococcal and streptococcal exotoxins are implicated in gram-positive shock. Although superantigen-related septic shock appears to be primarily mediated by tumor necrosis factor (TNF)-α and interleukin (IL) 12, the contribution of other cytokines cannot be discounted. (78, 79, 80).
The physiologic response to superantigens is similar to septic shock induced by gram-negative endotoxin (lipopolysaccharide, LPS). In fact, LPS and superantigens can work synergistically to produce lethal toxic shock. (81, 82, 83). Toxic shock syndrome can be exacerbated by the synergistic effects of TSST-1 with the SE/SPE family of toxins. (84, 85). Superantigen stimulation of immune cells can exacerbate autoimmune syndromes by causing the expansion of autoreactive T cell subsets, upregulation of MHC-II expression, and the potentiation of cytotoxic T cell response (86, 87, 88, 89, 90, 91).
Toxic shock syndrome is a specific syndrome caused by either the Stapylococcal or Streptococcal organisms. It is specifically caused by the toxins produced by these bacteria. Clinically it often occurs in young women and children and is characterized by a raised temperature, low blood pressure, a rash that eventually leads to skin loss especially on the palms and the soles and multi-organ involvement.
Septic Shock on the other hand involves both gram negative as well as gram positive organisms, occurs in all groups of patients especially the elderly and post-surgical. It has similar symptoms except for the lack of a skin losing rash. Both diseases have a high mortality-however there are many more cases of septic shock as compared to toxic shock. The term “septic shock” is used herein to describe hypotension and organ failure associated with bacterial infections.
“Toxic shock like syndrome” is the term previously used to describe the syndromes caused by staphyloccal and streptococcal pyrogenic bacterial exotoxins other than toxic shock syndrome toxin (TSST-1) from S. aureus. Currently, the term “toxic shock syndrome” is used to describe the syndromes caused by TSST-1 and the other pyrogenic exotoxins, and is the terminology used hereinafter.
Toxic shock syndrome can be exacerbated by the synergistic effects of TSST-1 with the enterotoxin/pyrogenic toxin family of toxins (9, 25). Gram negative bacterial endotoxin and the pyrogenic toxins can work synergistically to produce intractable shock (17, 30).
With respect to septic shock, lipopolysaccharide (LPS) is an integral part of the cell wall of Gram-negative bacteria and is a potent inducer of cytokine release by macrophages (52). During the induction phase of septicemia, LPS binds to the CD14 receptors of macrophages and triggers the release of a number of cytokines including Interleukin-1 (IL-1), and Tumor Necrosis Factor-α(TNF-α) (49). Accordingly, therapeutic strategies for septic shock have centered on the neutralization of LPS or LPS-induced cytokines (64). Unfortunately, trials using either monoclonal antibodies directed against part of the LPS molecule or the use of CD14 soluble receptors have not been very promising (45). The reasons for these failures might be: 1. The type of patient selected (many were already in irreversible shock). 2. The monoclonal antibody did not block all sites of LPS. 3. Soluble CD14 receptors did not block all LPS molecules.
Toxic shock syndrome and septic shock are still among the most life threatening syndromes affecting humans. It is estimated that approximately 20,000 cases of toxic shock syndrome occur each year of with a 10% mortality rate (66). With respect to septic shock approximately 400,000–500,000 cases occur each year with a 50% mortality (63). Present therapy is primarily symptomatic with administration of fluids, antibiotics, pressor agents and occasionally steroids (56). There is no vaccine available for toxic shock syndrome since all of the superantigens are antigenically distinct even though there is some sequence homology present in all the superantigens. There have been numerous vaccine trials for septic shock none of which have been successful.
With respect to the failed vaccine trials for septic shock, we believe that there was a failure to recognize that the interaction between the superantigens described above and LPS enhances the lethal potency of both these antigens by about 1000 fold. In contrast, each antigen when given alone requires a much higher dose for lethal septic shock (46).
Hence, it is proposed that at least two independent pathways of lethal septic shock can occur. LPS and peptidoglycan interact with macrophages. The superantigens interact with T cells. In both cases target cells are induced to release large amounts of cytokines. There is increasing evidence that gram-positive infections frequently accompany gram-negative infections in patients with septic shock (see article by Rangel-Frausto, pages 299–312) (96). Exposure to gram-negative endotoxin produces a state of macrophage hyperesponsiveness on subsequent stimulation (92). A similar state is seen with monocytes in septic shock. Our group and others have shown that LPS and superantigens can act synergistically to produce lethal septic shock in animal models (93). It is our hypothesis that a significant amount of septic shock involves an early gram-negative infection that causes significant symptoms of vasodilation and hypotension. This is then treated with fluids and antibiotics, leading to early recovery by the patient. Some days later, a gram-positive insult either via a line sepsis of the skin or gastrointestinal flora may cause severe irreversible shock in a previously LPS-sensitized patient. This model is depicted graphically in FIG. 2 herein (94).
In other words, since Gram-negative and Gram-positive organisms can be recovered from patients with sepsis, it appears that it is the “two hit” hypothesis that is operative and the interaction between LPS and the superantigens markedly enhances the lethal properties of both molecules. In this model, the interruption of the toxin pathway by anti-peptide antibody(ies) or by peptide(s) of the invention prevents the onset of lethal shock induced by the combination of the LPS and one or more of the superantigens.